Data Sheet
Contents
1. 4 Celeste A Fernandez Capetillo O Kruhlak MJ Pilch DR Staudt DW Leef A Bonner RF Bonner WM Nussenzweig A Histone H2AX phosphorylation is dispensable fo ial recognition of DNA breaks Nat Cell Biol 2003 Jul 5 7 675 9 5 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA histone H2AX phosphorylation on serine 139 J Biol Chem 1998 6 Rogakou EP Nieves Neira W Boon C Pommier Y Bonner W during apoptosis induces phosphorylation of H2AX histone 31 275 13 9390 5 e stranded breaks induce 3 10 5858 68 ation of DNA fragmentation 39 J Biol Chem 2000 Mar Related Products CycLex Cellular Histone Acetylation Assay Ki 40 CycLex Cellular UV DNA Damage Detection F at CY 1141 CycLex BrdU Cellular ELISA Kit Cat CY 114 CycLex Histone H2A X Phosphorylation Cellular K A Kit Cat CY 1143 CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 7 oducts are supplied for research use only CycLex CircuLex products and may not be resold modified for resale or used to manufacture commercial prior written approval from CycLex Co Ltd To inquire about licensing for Version 120420 CY 11432. Q Cells Well C CY 1143 10 Version 120420 Histone H2A X Phosphorylation Cellular ELISA Kit Yelex User s Manual A For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of Camptothecin on phosphorylation of histone H2A X in HeLa The cells 2 5 x 10 w were treated with indicated concentration of drugs for 1 hr Dose dependency of Camptothecine cA 2 0 1 8 ai Stas Fa ea 50000 cell well 25000 cell well wa 12500 cell well eee gt 6250 cell well lt A450 5 0 00 0 01 0 10 1 00 10 00 100 00 Camptothecin uM C CY 1143 11 Version 120420 p Histone H2A X Phosphorylation Cellular ELISA Kit f yel ex User s Manual For Research Use Only Not for use in diagnostic procedures References Burma S Chen BP Murphy M Kurimasa A Chen DJ ATM phosphorylates histone H2A response to DNA double strand breaks J Biol Chem 2001 Nov 9 276 45 42462 7 2 Bassing CH Suh H Ferguson DO Chua KF Manis J Eckersdorff M Gleason M Bro Alt FW Histone H2AX a dosage dependent suppressor of oncogenic translocations Cell 2003 Aug 8 114 3 359 70 3 Kobayashi J Tauchi H Sakamoto S Nakamura A Morishima K Matsuura S Kobayashi T Tamai K Tanimoto K Komatsu K NBS1 localizes to gamma H2AX foci through interaction with the FHA BRCT domain Curr Biol 2002 Oct 29 12 21 1846 51
3. research use only and not recommended for internal use in humans or anim A chemicals should be considered potentially hazardous and principles of good laboratory praetiee s d be followed Technical Notes When performing washes manually avoid introducing bubbles when dispensing li and ensure each well is filled with buffer but not overflowing to avoid cross co wells Empty wells with a wrist flick motion over an appropriate receptacle and blot any remaining moisture onto clean absorbent paper 2 Agitation of wells during incubation of Blocking Buffer and Antibody steps 1 ended to reduce non specific background If microtiter plate agitator is not available a ex at a low setting into the wells can be used e g level 1 of Fisher s Genie II platform vortex If back roblems occur simply increase the number and or duration of washes 3 A brief 1X PBS rinse is recommended prior to the addition of the of the Tween 20 with can interfere with the HRP activity Do not allow the wells to dry out during the protocol Incubation temperatures for Primary Antibody and Detectio tib can be varied and should be empirically determined ate to remove any traces ns General Notes e Allow all the components to come to room temp efore use e Do not use kit components beyond the indicated kit e tion date e Rinse all detergent residue from glassware Qy e Use deionized water of the highest quality e Do not mix reagents fr
4. wavelength can be used Wells must be read within 30 minutes of add the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After remove any remaining Wash Buffer by aspirating or decanting Invert the pla against clean paper towels Note 2 If the microplate reader is not capable of reading absorbance greater than t rbance of the highest standard perform a second reading at 405 nm A new standard cur ructed using the values measured at 405 nm is used to determine of histone H2A X ation level of off scale samples The readings at 405 nm should not replace the on s ings at 450 nm Troubleshooting j The signals are influenced a great deal by cell line and cell ensure the appropriate cell number for your experiment See at you plated please ple of Test Results Fig 2 2 Unavoidable background at Treatment time 0 hr or v appropriate cells number is used It is usually aroun Fig 1 rol is observed even if an Example of Test Results 0 cells well in case of adherent cells control background rather than those in 3 With some cell lines higher cell concentrations an may lead to increasing absorbance values i Camptothecin treatment 4 All treatments including treatment of Cam ecin should be run in duplicate using the protocol described in the Detailed Protocol Incu imes or temperatures significantly different from those specified may give erroneo
5. 10X Wash Buffer 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X ffer provided to 900 mL of deionized distilled water Mix well 2 95 MetOH For each 96 well plate add 1 mL H O to 19 mL of m ool in 20 C freezer This solution must be prepared fresh Discard unused portion follo completion 3 1 paraformaldehyde in PBS For each 96 well plate dissolve aformaldehyde in 20 mL of PBS pH7 2 This solution must be prepared fresh Dis ard u d portion following assay completion Assay Procedure A Culture adherent cells in 96 well microplate tment with compounds 1 Plate adherent cells in 96 well microplate at 1 5 x 10 cells well 2 Incubate the microplate at 37 C over night ncubator 3 Add appropriate amount of test compounds to each well Camptothecin treatment should be run in duplicate as a positive control for i histone H2A X phosphorylation Please include vehicle control e g H20 in case of Cam in 4 Incubate the microplate at of ropriate time Camptothecin treatme e cells with 20 uM Camptothecin for 0 0 25 0 5 1 2 3 4 and 6 ours C CY 1143 6 Version 120420 4 Histone H2A X Phosphorylation Cellular ELISA Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures B Fixing cells to 96 well microplate and blocking Single Fixation protocol Fixing of the cells in the 96 well plates should be done as soon as the desired treatment has c
6. 4 Histone H2A X Phosphorylation Cellular ELISA Kit cLex User s Manual For Research Use Only Not for use in diagnostic procedures Cell Based ELISA Kit for Measuring phosphorylation of Histone H2A X in situ CycLex Histone H2A X Phosphorylati Cellular ELISA Kit amp Cat CY 1143 Intended ESO sientctssetecsveraasciarialie daisy 1 Qy DAG Enneco i eaaeestie eas as 1 ntroduction s ssseeeeeeseeeessesssessrsssresesessessee 2 Principle of the Assay 2 3 Materials Provided is cscitccsssaststcactasaceasdedcivedes 4 Materials Required but not Provided 4 Precautions and Recommendations 5 Detailed Proto Gl scsi ccsssacsvcsansastectnasncadioiis 6 9 TrOuBleshO Ot iis cetiscsdiesiesiecasvamtareaesieceends 9 Reagent SCADILY siceresetavseccssastavaravecntveracisniseas 9 Example of Test Result ic sisstssscasssstsaicssecsees 10 11 Referen CS 32832 2e ec ftccct ceteeescdaestanteecoieiets 1 Related Products 1 Intended Use The CycLex Research Product Histone X Phosphorylation Cellular ELISA Kit is a non isotopic immunoassay used for th quantitative measurement of histone H2A X phosphorylation level in response to DNA nd break in situ by means of cell based ELISA Applications for this kit include 1 Monitoring the effects of phar agents on histone H2A X phosphorylation in cells 2 Study on the DNA double strand b repair mechanisms in situ 3 Screening compounds cause DNAdouble strand break in cells This a
7. al Substrate Reagent 20 mL of the chromogenic substrate tetra m 1 2504 containing 12 mL of eady to use ine TMB Ready to use Stop Solution One bottle supplied ready to use containing Materials Required but not Provid Cell culture flasks for growing and splitting cells Cell culture media Ice cold 95 Methanol for 1 fixation of cells 1 paraformaldehyde in PBS for 2 fixati ells 1X PBS pH 7 2 Pipettors 2 20 uL 20 200 uL and 200 ecision pipettors with disposable tips Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for s ation e Vortex mixer e Microplate washer optional Ma washing is possible but not preferable e Software package facilitati eration and analysis optional e 500 or 1000 mL graduate e Reagent reservoirs Deionized water of the est quality e Absorbent paper aper towels e Plate reader capa asuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wa g 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelen 450 nm which will give a somewhat higher reading amp Y C CY 1143 4 Version 120420 4 Histone H2A X Phosphorylation Cellular ELISA Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Safety Warnings and Precautions The Histone H2A X Phosphorylation Cellular ELISA designed for
8. ck While still inverted tap onto absorbent paper Incubate the plate for 1 hour at room temper microplate shaker 12 Remove Secondary Antibody Solution wi ist flick 13 Rinse wells once with 200 pL well W 14 Remove Wash Buffer with wrist flic plate onto absorbent paper 15 Wash wells 4 times with 200 on an orbital microplate sh we ash Buffer for 2 minutes each with shaking at ca 200 rpm ove Wash Buffer in between each wash with a wrist flick 16 After last wash with r rinse wells once with 300 pl well 1X PBS Remove with a wrist flick and tap on so t paper Ensure that that no liquid remains in the well 17 Add 50 pL well the plate wi after the nece e Reagent Avoid exposing the microplate to direct sunlight Covering um foil is recommended Return Substrate Reagent to 4 C immediately e is removed 18 Incubate r 10 20 minutes at room temperature ca 25 C shaking at ca 300 rpm on an gbital roplate shaker The incubation time may be extended up to 30 minutes if the reaction te r elow than 20 C Cat i CY 1143 8 Version 120420 Histone H2A X Phosphorylation Cellular ELISA Kit Pa G ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 20 Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelengt of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the micro at 450 nm if only a single
9. om different kits e The buffers and reagents used in kit Contain NaN as preservatives Care should be taken to avoid direct contact with these rea e Dispose of tetra methylbenizi B containing solutions in compliance with local regulations e Avoid contact with t i top Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves ction when handling immunodiagnostic materials and samples of human origin and the ents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroug 1 ater and seek medical attention when necessary ic Acid is a strong acid Wear disposable gloves and eye protection when dling Stop Solution CY 1143 Version 120420 Histone H2A X Phosphorylation Cellular ELISA Kit e cLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product Histone H2A X Phosphorylation Cellular ELISA Kit includ reagents except cell fixative Since experimental conditions may vary treatment cells with Campt within the kit should be included in each experiment as a positive control for inductio H2A X phosphorylation Disposable pipette tips and reagent troughs should be used iq transfers to avoid cross contamination of reagents or samples Preparation of Reagents All reagents need to be brought to room temperature prior to the assay Assay r are supplied ready to use with the exception of
10. omplet 1 Remove media from wells with a wrist flick Avoid touching the bottom of the well and ing cells 2 Immediately add 150 pL well of 95 MetOH as a fixative slowly to insure cells ar hed from the plastic Let stand for 10 minutes at room temperature ca 25 C w Remove 95 MetOH from wells with a wrist flick While still inverted tap t e gently onto absorbent paper to remove any excess fixing agent still within the wells A Add 200 uL well Wash Buffer Let stand for 1 minute at room temperatu e 5 Remove wash buffer with a wrist flick While still inverted gently tap nto absorbent paper to remove any excess liquid 6 Add 200 uL well Blocking Reagent and incubate for 2 hours at overnight at 4 C Alternatively In cases where the cells are easy to detach from lp even aftter1st fixation B Fixing cells to 96 well microplate and blocking Do ion protocol 1 Remove media from wells with a wrist flick i Pid Duching the bottom of the well and removing cells 2 Immediately add 150 uL well of 95 MetOH as a fixative slowly to insure cells are not detached from the plastic Let stand for 10 minutes Q temperature ca 25 C 3 Remove 95 MetOH from wells wi ick While still inverted tap the plate gently onto absorbent paper to remove any excess fixingfagent still within the wells 4 Add 150 pL well of 1 paraf de in PBS slowly to ensure cells are not dislodged f
11. ppropriate time at 37 C in CO in Discard the culture medium and wash the microplate D Add 150 uL of ice cold 95 methanol for fixation v Stand for 10 min at room te Discard the methanol Y Add 200 uL of Blocking Reagent y Incubate for atleaf 2 hr a037 C or O N at 4 C Add 50 uL of Anti Phospho Histone H 39 Monoclonal Antibody 4 Incubate 1 Wash fi wells oom temp Add 50 uL of y conjugated Cs hr at room temp Wash the wells Add 50 uL of Oj Add 50 uL of S O Meas ce at 450 nm C CY 1143 3 Version 120420 Histone H2A X Phosphorylation Cellular ELISA Kit Pa G ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All compounds treatment and positive control Camptothecin treatment should be assay duplicate The following components are supplied and are sufficient for the two 96 well microplat Microplate Two 96 well cell culture plates 100X Camptothecin One vial containing 50 uL of 2 mM Camptothecin in DMSO 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 Blocking Reagent Two bottles containing 20 mL of 1X Blocking Reagent Read Q Primary Antibody Solution Anti Phospho Histone H2A X S139 Monocl ibody One vial containing 12 mL of anti phospho histone H2A X S139 monoclonal antib 1 Ready to use Secondary Antibody Solution HRP conjugated Anti Mouse IgG HRP horseradish peroxidase conjugated anti mouse IgG polyclon
12. rom the wells Let stand for 5 minu temperature ca 25 C 5 Remove paraformaldehyde g on from wells with a wrist flick While still inverted gently tap the plate onto absorbent pap 2move any excess liquid still in the wells a wrist flick While still inverted gently tap the plate onto absorbent paper d 6 Add 200 pL well Wash er Let stand for 1 minute at room temperature ca 25 C T Remove was 1 to remove any S 1 8 Add 200 cking Reagent and incubate for 2 hours at 37 C or overnight at 4 C C CY 1143 T Version 120420 p Histone H2A X Phosphorylation Cellular ELISA Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures C Detection of Signals Addition of Primary and Secondary Antibodies and Substrate Reagent p Remove Blocking Reagent with a wrist flick 2 Rinse the wells once with 200 uL well of Wash Buffer This can be achieved either b multichannel pipette or a manifold 3 Remove Wash Buffer with a wrist flick While the plate is still inverted tap onto absorbe remove any excess buffer within the wells 4 Add 50 uL well of Primary Antibody Solution into each well 5 Incubate the plate for 1 hour at room temperature ca 25 C shaking at cag80 on_an orbital microplate shaker 6 Remove Primary Antibody Solution with a wrist flick 7 Rinse the wells once with 200u1 well of Wash Buffer Ge Remove Wash Buffer with a wrist fli
13. ssay kit is for resear g ofly and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store C CY 1143 1 Version 120420 4 Histone H2A X Phosphorylation Cellular ELISA Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Histone H2A X has been implicated in the maintenance of genomic stability in response to double strand breaks DSBs It is phosphorylated at an evolutionary conserved PI3 kinase related Kinas motif in the carboxyl terminus within seconds after exposure to ionizing radiationga R Immunofluorescence studies have revealed that phosphorylated H2AX y H2AX forms 1 the sites of DSBs These foci appear within min after exposure of cells to IR Their nur in the first 10 30 min after irradiation before they gradually decline correlating with the pi of slowly re joining DSBs 2 y H2AX foci are also found at sites of V D J recombinatio DSBs in developing thymocytes 3 and at sites of recombinational DSBs during meigsis 4 In addition phosphorylation of H2AX is also induced by initiation of DNA fragmentation during ApS ptosis 5 Thus H2AxX is phosphorylated in response to DSBs Principle of the Assay The CycLex Histone H2A X Phosphorylation Cellular ELISA Kit base histone H2A X at serine139 in response to ionizing radiation or DNA DNA double strand break in cells that are cultured in microtiter plate During trea
14. tment of genotoxic reagent is carried out on wells o otiter plate histone H2A X will be phosphorylated at serine 139 in the living cells e antibody binding to the phosphorylated histone H2A X cells must be fixed and perm abilized Detector anti phospho histone H2A X S139 monoclonal antibody clone T 2F1 is pipette wells and allowed to incubate for one hour during which time it binds to any the phosphogylat istone H2A X Unbound antibody is washed away and horseradish peroxidase conjugatedg oa i se IgG is added which binds to the version of the chromogenic substrate a blue solution or yellow after the addition of hotometry and reflects the relative amount of phosphorylation of the g reagent which cause tetra methylbenzidine TMB from a colorless solut stopping reagent The color is quantified by spect phosphorylated histone H2A X in the cells C CY 1143 2 Version 120420 wn Histone H2A X Phosphorylation Cellular ELISA Kit Ce yCLex User s Manual For Research Use Only Not for use in diagnostic procedures The CycLex Histone H2A X Phosphorylation Cellular ELISA Kit is designed to measure relative levels of phosphorylated histone H2A X in situ The summary of the assay is shown in below Summary of Procedure for adherent cells C cA Culture adherent cells in microplate at 1 5 x 10 cells well v Incubate O N at 37 C in CO incubator Add appropriate amount of test compound for induction of DNA double st Yy Incubate a
15. us results 5 Poor duplicates accompanied by el yated values for wells containing non treated cells vehicle control indicate insufficient washi igorous washing Wash the plate thoroughly and gently 6 Overall low signal may indicate desiccation of the plate has occurred between the final wash and addition of Substrate Re not allow the plate to dry out Add Substrate Reagent immediately after wash All of the reagen ed in the CycLex Research Product Histone H2A X phosphorylation Cellular ELISA een tested for stability Reagents should not be used beyond the stated expiration date eceipt kit reagents should be stored at 4 C For research not for use in diagnostic or therapeutic procedures Cy 1143 Version 120420 Histone H2A X Phosphorylation Cellular ELISA Kit cA yCLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Typical result of time course experiment using HeLa cells treated with 10 uM SN38 AN 25 000cells wel 50 000 cells well 0 9 12 08 1 0 07 08 0 6 O g 06 T lt lt 04 03 0 4 0 2 0 2 0 1 0 0 09 0 1 2 3 4 5 0 1 2 3 4 5 SN38 Treatment Time hr SN38 Treatment Time hr gt Fig 2 Effect of cell number on ELISA value using HeLa c 10 uM SN38 0 12 500 25 000 50 000
Download Pdf Manuals
Related Search